Toxicokinetics of p-tert-octylphenol in male and female Sprague-Dawley rats after intravenous, oral, or subcutaneous exposures.

This study was undertaken to characterize the toxicokinetics of p-tert-octylphenol (OP), a weak estrogenic compound, in male and female rats. Male and female Sprague-Dawley rats were given a single dose of OP either by oral gavage (50, 125 or 250 mg/kg), by intravenous (iv) injection (2, 4, or 8 mg/kg), or by subcutaneous (sc) injection (125 mg/kg). In a repeated dosing experiment, rats were given OP (oral) daily (25, 50, or 125 mg/kg) for 35 d (female) or 60 d (male). Blood and tissue samples were collected and analyzed for OP content using gas chromatography with detection by mass spectrometry. Blood OP concentrations were generally higher in female than male rats following a single oral or sc administration but were similar following a single iv injection. Tissue OP concentrations were also higher in female than male rats following oral exposure, consistent with the faster metabolism of OP observed in male rat liver microsomes. After subchronic administration, blood OP concentrations were higher at the end of exposure for female (33 d) (2.26-fold, not significant) and male (57 d) (3.47-fold) rats than single dosing but there was no change in the tissue OP concentrations. Gender differences in tissue OP concentrations may contribute, in part, to gender differences in the toxicity of OP in rats. The fact that OP was found in all reproductive tissues confirms its potential for direct endocrine-like effects.

Determination of p-tert-octylphenol in blood and tissues by gas chromatography coupled with mass spectrometry.

A sensitive and reproducible procedure using gas chromatography coupled with mass spectrometry is described for the determination of p-tert-octylphenol (OP), a persistent degradation product of alkylphenol ethoxylates that binds to the estrogen receptor in blood and tissues. The first step involved the extraction of blood (200 microL) or tissue homogenate (400 microL) with methyl tert-butyl ether, including p-tert-butylphenol (BP) as internal standard. After extraction, the sample was evaporated to dryness with a gentle stream of nitrogen at 45 degrees C, and OP and BP were derivatized with an acetylation reaction involving acetic anhydride and catalyzed by pyridine. Samples were then analyzed by a gas chromatograph equipped with a mass spectrometer (single ion monitoring) with a Varian VF-5ms capillary column. The limit of detection and the limit of quantification of the method in blood were 4.6 and 15.5 ng/mL, respectively. The linearity and reproducibility of the method were acceptable, with coefficients of variation of approximately 10% for blood and ranging between 9% and 27% for tissues. This method was applied to the determination of unchanged OP in blood and tissues obtained from Sprague-Dawley rats after oral and IV OP administration.

In vitro ovarian tissue and organ culture: a review.

Many investigations have utilized techniques for culturing ovarian tissue or isolated follicles in vitro. Whole ovaries from fetal or neonatal rodents have been incubated in organ culture systems. This has been utilized to understand the sequence of follicle formation and its hormonal requirements, activation of quiescent follicles, follicular growth and development, and acquisition of steroidogenic capabilities. Adaptations of this technique include incubation of ovaries in a chamber continuously perfused with medium or perfusion of medium through the intact vasculature. Late follicular development, ovulation, and steroidogenesis can also be examined in these systems. Another approach has been to culture individual follicles isolated by enzymatic or mechanical dissociation. The majority of this research has focused on improving follicular development in vitro. This review will discuss these various culture techniques and some of the results that have been acquired. Recent results from toxicological studies utilizing whole ovarian cultures performed will also be described. Future applications for ovarian and follicular cultures may include in vitro follicular development for eventual production of offspring from frozen ovarian tissue, mechanistic studies related to the impact of endocrine disruptors and ovotoxicants on ovarian function, and further investigations into follicle activation and development.

Effect of 4-vinylcyclohexene diepoxide dosing in rats on GSH levels in liver and ovaries.

Repeated daily dosing of rats with the occupational chemical 4- vinylcyclohexene or its diepoxide metabolite (VCD) for 15 days destroys the smallest ovarian follicles. VCD acutely reduced hepatic levels of the antioxidant, glutathione (GSH); therefore, these studies were designed to evaluate whether GSH concentrations mediate VCD-induced ovotoxicity. Immature female Fischer 344 rats were dosed once or daily for 15 days with VCD (0.57 mmol/kg, ip) or the GSH synthesis inhibitor buthionine sulfoximine (BSO, 2 mmol/kg, ip). Animals were euthanized 2, 6, or 26 h following a single dose, and 2 or 26 h following 15 days of daily dosing. Reduced (p < 0.05) hepatic GSH was seen within 2 h of a single dose of either VCD (51 +/- 5% of control) or BSO (42 +/- 9%), but only BSO reduced ovarian GSH (71 +/- 5% at 6 h, p = 0.05) as measured by HPLC. Within 26 h, GSH levels had returned to control levels with either treatment. Hepatic GSH levels were reduced (< 0.05) 2 h after 15 daily doses with BSO (42 +/- 5%) or VCD (70 +/- 4%), but only BSO decreased ovarian GSH (64 +/- 3%). GSH levels in 15-day tissues were similar to controls 26 h after the final dose. Neither BSO nor VCD increased hepatic or ovarian concentrations of the oxidized dimer of GSH (GSSG) or thiobarbituric acid-reactive substances (TBARS), indicators of oxidative stress. These results suggest these treatments did not cause an oxidative stress. Histological counts of ovarian small follicle numbers were reduced (p < 0.05) in 15-day VCD-treated rats, whereas BSO did not affect follicle numbers, even though BSO reduced ovarian GSH content. These results support the conclusion that alterations in ovarian GSH levels are not involved in VCD-induced ovotoxicity.

Ovarian toxicity of 4-vinylcyclohexene diepoxide: a mechanistic model.

Female mammals are born with a finite number of ovarian primordial follicles that cannot be regenerated; thus, chemicals that destroy oocytes contained in these follicles can produce premature ovarian failure (early menopuase in women). Exposure of women to known ovotoxicants, such as contaminants in cigarette smoke, is associated with early menopause. Thus, the potential risks posed by ovotoxic chemicals is of concern. Our studies have focused on the environmental chemical 4-vinylcyclohexene (VCH), which is produced during the manufacture of rubber tires, flame retardants, insecticides, plasticizers, and antioxidants. Dosing of female rats and mice with the ovotoxic diepoxide metabolite of VCH, 4-vinylcyclohexene diepoxide (VCD), for 30 days destroyed the majority of ovarian primordial follicles. Using VCD in rats as a generalized model for ovotoxicity, we determined that 1) repeated daily dosing is required, 2) cell death is via apoptosis, and 3) altered expression of specific genes is involved. An integrated approach at the morphologic, biochemical, and molecular level was used to support these conclusions. Studies in isolated rat small preantral follicles (targeted for VCD-induced ovotoxicity) focused on the role of cell death genes, mitochondrion-associated events, and VCD metabolism. We also evaluated how this information relates to human risk for early menopause. These animal research results provide a better understanding of the potential risk of human exposure to environmental ovarian toxicants and greater insight as to the impact of these toxicants on reproductive health in women.

Ultrastructural evaluation of oocytes during atresia in rat ovarian follicles.

Mammalian females are born with a finite number of ovarian oocytes, the vast majority of which ultimately undergo degeneration by atresia. The overall process of ovarian follicular atresia has been morphologically well described only in large antral follicles. Additionally, little attention has been focused on ultrastructural changes in the oocyte. Furthermore, most such morphological studies were performed prior to identification of apoptosis as a mechanism of physiological cell death. Therefore, the purpose of this study was to use electron microscopy to compare the process of atretic oocyte degradation in ovarian follicles of female Fischer 344 rats (38 days old) with ultrastructural characteristics of apoptosis. Examination of ovarian follicles revealed that nucleolar segregation, cytoplasmic or nuclear condensation, apoptotic body formation, and chromatin margination along the nuclear membrane are never observed in atretic oocytes during the degenerative process. Instead, early morphological changes in atretic oocytes include retraction of granulosa cell- and oocyte-derived microvilli and condensation of mitochondria and loss of cristae. These occurrences coincide with initiation of granulosa cell apoptosis. After most granulosa cells are lost, more severe changes occur, including segmentation of the oocyte and cytoplasmic vacuolization as atresia progresses. Thus, these results suggest that, during atresia, oocytes are removed by physiological oocyte cell death, a method that does not involve classically described apoptosis.

A fiber-optic cocaine biosensor.

A fiber-optic biosensor was developed for detection of cocaine, its metabolites, and other coca alkaloids, using a monoclonal antibody (mAb) against a derivatized benzoylecgonine (BE). The mAb was immobilized noncovalently on quartz fibers and a flow fluorometer was used to detect changes in evanescent wave fluorescence. A fluorescein (FL) conjugate of BE bound to the mAb specifically in a saturable manner and with high affinity (Kd = 7.6 nM). Cocaine or other test compounds competed with FL-BE for binding to the mAb in a concentration-dependent manner, thereby reducing the initial rate or steady-state fluorescence. Addition of cocaine to the flow buffer after reaching steady-state fluorescence enhanced the dissociation of bound FL-BE, and cocaine removal allowed fiber regeneration for multiple measurements. The detection limits for cocaine, cocaethylene, norcocaine, and BE were 5, 5, 29, and 30 ng/ml, respectively, but for ecgonine it was 4600 ng/ml and for methylecgonine it was 2000 ng/ml. Tropacocaine was detected at 10 ng/ml, but atropine was detected at 2900 ng/ml. The biosensor discriminated by 833-fold between cocaine and its stereoisomer pseudococaine. Structural features necessary for high-affinity recognition by this mAb are benzoate and 3 beta configuration, both of which are found in BE, cocaine, norcocaine, and cocaethylene.

Corporate-sponsored breast cancer screening at the work site: results of a statewide program.

Mobile screening mammography was offered to 3,627 employees of a large corporation in Pennsylvania and Delaware. The examination was available to women employees or employee spouses aged 35 years and older. Women were charged $30 for a standard two-view examination. They also received health education materials on mammography and breast self-examination. The remaining costs of the program were underwritten by the corporation. During this program, 3,627 mammographic studies were performed; 63 biopsies were recommended. Fifty-seven biopsies were performed, and nine cancers were diagnosed. Costs of this program are presented in detail. The authors conclude that mobile screening programs at the work site provide an inexpensive, convenient alternative to more traditional screening programs. The inherent advantages of this program are the low cost, the relative ease with which the examination can be performed, and the positive role that corporate medical personnel assume in encouraging individual and group participation.

Evaluation of Nitrate Reductase Activity in Rhizobium japonicum.

Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase.